Phase separation after chloroform addition was not particularly good. Aqueous phases in sample 424 was a bit cloudy (salty?) with no defined interphase. The remaining two samples did exhibit a defined interphase and were the aqueous phases were less cloudy than sample 424, but were far from ideal.

Quantified RNA using Roberts Lab Qubit 3.0 with the Qubit RNA high sensitivity kit. Used 5uL of each sample.

RESULTS

No detectable RNA in any samples. Samples were discarded.

As has been the case for all samples in this project, RNA isolation methodologies have produced wildly inconsistent results.

Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.

I’ve set both Emu & Roadrunner R Studio Server to use this installation of R by changing the /etc/restudio/rserver.conf file to the following:

These two bits of information led me to believe the problem wasn’t that the system upgrade to 18.04 was incompatible with these old Apple Xserve hardware (since the upgrade didn’t actually get implemented) and instead was that the upgrade might have been initiated, but aborted, which modified the GRUB configuration file(s), breaking the GUI; much like the problem I previously addressed earlier this summer.

When I fixed the display/GUI issues with Emu and Roadrunner earlier this summer, I noted that the /etc/default/grub files on each of the computers were slightly different, despite the fact that these two computers should be identical. So, I replaced the /etc/default/grub file on Emu with the file from Roadrunner and rebooted Emu.

Contents of /etc/default/grub file on Emu/Roadrunner, for future reference:

# If you change this file, run 'update-grub' afterwards to update
# /boot/grub/grub.cfg.
# For full documentation of the options in this file, see:
# info -f grub -n 'Simple configuration'
GRUB_DEFAULT=0
#GRUB_HIDDEN_TIMEOUT=0
GRUB_HIDDEN_TIMEOUT_QUIET=true
GRUB_TIMEOUT=10
GRUB_DISTRIBUTOR=`lsb_release -i -s 2> /dev/null || echo Debian`
GRUB_CMDLINE_LINUX_DEFAULT="quiet splash"
GRUB_CMDLINE_LINUX=""
# Uncomment to enable BadRAM filtering, modify to suit your needs
# This works with Linux (no patch required) and with any kernel that obtains
# the memory map information from GRUB (GNU Mach, kernel of FreeBSD ...)
#GRUB_BADRAM="0x01234567,0xfefefefe,0x89abcdef,0xefefefef"
# Uncomment to disable graphical terminal (grub-pc only)
#GRUB_TERMINAL=console
# The resolution used on graphical terminal
# note that you can use only modes which your graphic card supports via VBE
# you can see them in real GRUB with the command `vbeinfo'
#GRUB_GFXMODE=640x480
# Uncomment if you don't want GRUB to pass "root=UUID=xxx" parameter to Linux
#GRUB_DISABLE_LINUX_UUID=true
# Uncomment to disable generation of recovery mode menu entries
#GRUB_DISABLE_RECOVERY="true"
# Uncomment to get a beep at grub start
#GRUB_INIT_TUNE="480 440 1"

I decided that I wanted to use the Olurida_v080 version instead (or, in addtion to?), as the Olurida_v080 version has not been size restricted (the Olurida v081 version is only contigs >1000bp). I feel like we could miss some important regions, so wanted to run this analysis using all of the genome data we currently have available. Additionally, this will be consistent with my previous Bismark (DNA methylation analysis).

Yesterday’s attempt at producing a bedgraph was a failure and a prodcuct of a major brain fart. The worst part is that I was questioning what I was doing the entire time, but still went through with the process! Yeesh!

The problem was that I tried to take our Trinity-assembled transcriptome and somehow align that to our genome. This can’t work because each of those assemblies don’t know the coordinates used by the other. So, as was the case, you end up with a bedgraph that shows zero coverage for all genome contigs.

I’d expect multiple entries for each contig (ideally), indicating start/stop positions for where transcripts align within a given contig. However, this appears to simply be a list of all the genome contigs and their lengths (Start=0, Stop=n).